Retroviruses are a family of viruses that contain an RNA genome and reverse transcriptase activity. Many classes of retroviruses have been identified, one of the major classes being the T-lymphotropic viruses which include HTLV-I which appears to be causative of adult T-cell leukemia-lymphoma; HTLV-II which appears to be related to a variant of hairy cell leukemia; and HIV-1 (HTLV-III) and HIV-2 which are linked to Acquired Immune Deficiency Syndrome (AIDS). Patients that test positive for HIV may be initially asymptomatic but typically develop AIDS related complex (ARC) followed by AIDS. The AIDS virus attacks and depletes the T lymphocytes causing severe immunosuppression in infected individuals which predisposes them to debilitating and ultimately fatal opportunistic infections.
No treatment is currently available to prevent or reverse the immunodeficiency of AIDS and ARC caused by the HIV virus. However, a number of compounds that interfere with viral replication have been developed to treat AIDS, including inhibitors of HIV reverse transcriptase (RT), such as nucleoside analogs including 3'-azido-3'-deoxythymidine (AZT, zidovidine) and 2',3'-dideoxy nucleosides such as 2', 3'-dideoxyinosine (DDI, ddI) and 2',3'-dideoxycytidine )DDC, ddC).
AZT is in use for early treatment of HIV positive individuals as well as individuals with ARC and AIDS. Unfortunately, AZT is frequently toxic, causing bone marrow suppression resulting in anemia leucopenia and thrombocytopenia. Also, AZT-resistant HIV strains have been observed after six months of AZT treatment. DDI and DDC have been approved for treatment of individuals with ARC and AIDS only in combination with AZT.
Consequently, the search for effective and non-toxic treatments for AIDS continues. Due to the toxicity associated with synthetic compounds, some groups are looking to natural sources in the search for a treatment for AIDS.
Some natural compounds have been reported to show anti-HIV activity. One compound is glycyrrhizin which is isolated from the aqueous extract of licorice root (Glycyrrhiza radix)1!, and is known as an anti-inflammatory substance in Chinese medicine. This compound consists of one molecule of glycyrrhetinic acid and two molecules of glucuronic acid. Ito et al 1! investigated its antiviral action on the human immunodeficiency virus in vitro, using cytopathic effect and plaque forming assay system in MT-4 cells. Cloned Molt-4 cells, which are sensitive to HIV and fused to giant cells after infection, were used as a parameter for cytopathic effect of HIV, Glycyrrhizin inhibited HIV-induced plaque formation in MT-4 cells at a concentration of 0.6 mM, the 50% inhibitory dose being 0.15 mM. Glycyrrhizin inhibited the cytopathic effect of HIV and HIV-specific antigen expression in MT-4 cells at a concentration of 0.3 and 0.6 mM, respectively. Furthermore, glycyrrhizin inhibited giant cell formation of HIV-infected Molt-4 clone No. 8 cells.
Wang et al 2! have reported that fifteen novel non-peptide HIV-1 protease inhibitors were identified by flexible 3D database pharmacophore searching of NCI DIS 3D database. The pharmacophores they used in the search were derived directly from the X-ray determined structure of protease-inhibitor complexes. These 15 inhibitors, belonging to nine different chemical classes, are promising leads for further development. The two best inhibitors found, the 4-hydroxycoumarin dimer and the 2-hydroxynapthoquinone tetramer, had ID.sub.50 values of 0.32 and 0.75 .mu.M, respectively, for HIV-1 protease inhibition.
Ardisia japonica (Thunb.) B1. (Myrsinaceae), in the form of a decoction of the roots, is widely used in traditional Chinese medicine to stop cough and uterine bleeding. In vitro anti-HIV activity studies, norbergenin and bergenin were found with moderate anti-HIV activity 3!. The most active compound was norgergenin, which exhibited an EC.sub.50 value of 20 (.mu.g/ml) and a IC.sub.50 value of over 500 (.mu.g/ml) with a selectivity index above 25.
From Chrysanthemum morifolium Ramar (Compositae), acacetin-7-O-.beta.-D-galactopyranoside 4! was found to show significant anti-HIV activity. It has IC.sub.50 37 .mu.M, EC.sub.50 8 .mu.M and therapeutic index 5. Fujioka et al 5! found the MeOH extract of the leaves of Syzigium claviflorum (Roxb.) WALL (Myrtaceae) to show significant anti-HIV activity. Subsequent bioactivity-directed fractionation has resulted in the isolation of betulinic acid, platanic acid and dihydrobetulinic acid. These compounds were also potent inhibitors of HIV replication.
Geum is a genus of 65 species of rhizomatous herbs and subshrubs with simple or pinnately lobed leaves and regular flowers such as G. borisii, G. chiloense, G. coccineum, G. macrophyllum, G. montanum, G. reptans, G. rivale, G. triflorum, G. urbanum and G. japonicum etc.
Geum japonicum Thunb. is a perennial herb and the flowering plant of the Rosaceae family. The whole plant of Geum japonicum Thunb. has been used as a diuretic in traditional Chinese medicine 6!. The plants of Geum species are known to be rich in tannins. Several hydrolyzable tannins such as gemin A, gemin B, gemin C, gemin D, gemin E and gemin F have been isolated from Geum japonicum 7,8!. In the continuing investigation of the plant, some triterpenoids including 2-hydroxyoleanolic acid, 2-hydroxyursolic acid, 2,19-dihydroxy-ursolic acid, 2,3,19,23-tetrahydroxyurs-12-en-28-oic acid 28-O-D-glucopyranoside were isolated by Shigenaga et al 9!. Xu et al 10! isolated geponin, 1,2,3-tri-O-galloyl-D-glucopyranside, blumenol A, gallic aldehyde, 3,4-dihydroxybenzoic acid, caffeic acid and kampferol-3-O-glucopyranside from G. japonicum aqueous extract. Of these isolated compounds, geponin and gallic aldehyde showed strong anti-HSV activity.
One potential target for inhibiting the HIV virus is to inhibit the aspartyl protease that is encoded for by HIV(HIV PR). The function of the HIV PR is essential for proper virion assembly and maturation. Inactivation of HIV PR by either mutation or chemical inhibition leads to the production of immature, non-infectious viral particles 11,12!. HIV PR inhibitors represent a new class of therapeutic agents that complements existing approaches to antiviral therapy that targets another enzyme, the HIV reverse transcriptase, as discussed above.
To date, most inhibitors of the HIV aspartic acid protease have been transition state mimetics. These have included reduced amides (Moore et al., Biochem. Biophys. Res. Commun. 159:420-425 (1989); Dreyer et al., Proc. Natl. Acad. Sci. 86:9752-9756 (1989)), hydroxyethylene isoteres (Dreyer et al., Proc. Natl. Acad. Sci. 86:9752-9756 (1989); Vacca et al., J. Med. Chem. 34:1225-1228 (1991); Tomasselli et al., J. Biol. Chem. 265:14675-14683 (1990); Roberts et al., Science 248:358-361 (1990)), statine analogs (Dryer et al., 1989), phosphinic acid derivatives (Grobelny et al., Biochem. Biophys, Res. Commun. 169:1111-1116 (1990)) and difluoroketone derivatives (Dreyer et al., 1989; Sham et al., Biochem Biophys. Res. Commun. 175:914-916 (1991)).
Some natural compounds have been reported to be inhibitory to HIV-1 protease 13!. Thus, traditional medicines utilizing natural products appear to be useful sources for discovery of new anti-AIDS agents.